Neurons communicate with each other in the brain through specialized junctions, called synapses. During brain development, numerous new synapses are established and new synapses continue to form throughout life. The long-term goal of the research proposed in this application is to determine the molecular basis of synapse formation in the vertebrate brain. The first proteins have now been identified that organize synapse formation and development. One such protein is SynCAM 1, a synaptic cell adhesion molecule that connects pre- and postsynaptic sides. Importantly, SynCAM 1 induces the formation of new, fully functional excitatory synapses between neurons. It is highly expressed in the developing brain during intense synaptogenesis, indicating a broad function for this molecule in synapse formation. Such synaptogenic functions have been validated in cultured neurons and in vivo. The objective of this application is to define the signaling pathways through which SynCAM 1 organizes synapses and determine how other trans-synaptic proteins act in concert with it. The central hypothesis of this application is that SynCAM signaling organizes developing synapses and regulates synaptic function at later stages. To attain the objective of this application, three specific aims will be pursued. The first aim of this application is to determine the intracellular signaling pathways through which SynCAM 1- mediated synaptic adhesion instructs synapse development, focusing on changes in the synaptic cytoskeleton. Second, it will be analyzed how trans-synaptic interactions act in concert to assemble synapses and shape their structure. Third, it will be determined to which extent SynCAM 1 functions in vivo together with other synaptic adhesion molecules to organize synapses. These experiments involve the biochemical characterization of SynCAM binding partners and their activities. Functional analyses of SynCAM interactions will be performed by quantitative immunocytochemistry, imaging of synapses in cultured hippocampal neurons, and electrophysiological recordings. In addition, the in vivo relevance of these interactions will be tested using structural and functional studies of synapses, including ultrastructural analyses, electrophysiological recordings, and behavioral analyses. Achieving these goals is important for human health, as altered synapse organization affects the wiring of neuronal circuits and synaptic plasticity. These changes are associated with alterations in human behavior, the ability to learn and remember, and addiction to drugs of abuse. Furthermore, deficits of synapse formation likely underlie neurodevelopmental disorders such as autism. In summary, this application aims to identify the molecular interactions involved in synapse formation. The progress under this application will allow testing to which extent these synapse-organizing processes are affected in disorders of the human brain and whether they represent novel points of therapeutic intervention.